First case of a reversed Parabothus taiwanensis Amaoka & Shen, 1993 from Taiwan (Pleuronectiformes: Bothidae), with first evidence of situs inversus viscerum in bothid

Background Reversed condition is rarely found in most flatfishes in natural environment, except for some certain species. The mechanism controlling the reversals in flatfishes has been studied in some cultivated species, whereas some have only few cases for the entire family and remain unclear. Here, we report the first record of a dextral (reversed) specimen of Parabothus taiwanensis Amaoka & Shen, 1993 collected off southwestern Taiwan recently. It represents the second reversed case ever recorded in Bothidae. We aim to provide a detailed description of this dextral specimen and compared to the sinistral (normal) specimens collected from the vicinity. Methods Specimens were fixed in 4% formaldehyde and transferred to 70% ethanol for preservation. Meristic and morphometric characters were examined for both dextral and sinistral specimens. Dissections were made on specimens to confirm the position of internal organs. Lastly, X-radiographs were taken to elucidate the osteological features. Results As a result, no differences of both meristic and morphometric characters were observed between the dextral and sinistral specimens. Nevertheless, situs inversus viscerum is discovered in the dextral specimen for the first time in Bothidae and the sixth record within flatfishes.


INTRODUCTION
The sinistral flatfish family Bothidae, currently comprised of 20 genera and 169 species, are widely distributed in tropical to temperate waters globally (Amaoka & Ho, 2019;Fricke, Eschmeyer & van der Laan, 2023).Among them, the genus Parabothus (Norman, 1931) can be discriminated from other bothid genera by having an elliptical body; tip of isthmus below or slightly behind the posterior margin of the eye; scales on ocular side with moderately long ctenii; eyes separated by a concave space; usually absence of rostral spine in males; no white blotches on anterior margin of head (Amaoka, Mihara & Rivaton, 1997;Amaoka & Ho, 2019).
Larval flatfishes resemble typical fishes which have a bilaterally symmetrical body form and swim in an upright position in the water (Schreiber, 2006).These larvae undergo metamorphosis into juvenile (immature adult) form.During this process, one of the eyes migrates to the opposite side of the head and transforms into a lateralized swimming posture (Schreiber, 2006).Some flatfish species, for example, Paralichthys lethostigma and Psettodes erumei, can metamorphose into either sinistral or dextral morphology and behavior (Hensley, 2001;Schreiber, 2013).Nonetheless, among approximately 715 known species of flatfishes, only seven exhibit variations in dextral to sinistral polymorphism within geographically distinct populations, while the rest appear to be consistent on one side.Moreover, the exact selective advantage of sinistral versus dextral form still remains unclear (Schreiber, 2013).
Recently, an unusual dextral flatfish specimen was collected from the Ke-Tzu-Liao fishing port, southwestern Taiwan.After a close examination, this specimen is confirmed as a reversed form of Parabothus taiwanensis Amaoka & Shen, 1993.Examination of the same species deposited in the collection reveals that it is the only reversed specimen found in this species, and also the second specimen in the family.Moreover, the situs inversus viscerum is also discovered in our specimen which represents the first case of situs inversus viscerum in the Bothidae, and the sixth record in flatfishes.Here we describe the dextral specimen, compare it to conspecific sinistral specimens, and document the first finding of situs inversus viscerum within Bothidae in history.Hubbs & Lagler (1974) and Amaoka, Mihara & Rivaton (1993).Numbers of caudal-fin rays are expressed as upper unbranched ray + branched ray + lower unbranched ray.Gill rakers were counted on the outer face of the first ocular-side gill arch, with the raker at angle included in the lower-raker count.Scale pockets were adopted when scales were missing due to bottom-trawl operation.The vertebral formulae were determined by a digital X-ray machine (Dexela CMOS X-ray detector, Model 2315, Dexeal Co. Ltd., UK) set up in the National Museum of Marine Biology and Aquarium, Pingtung, Taiwan.Measurements were made using digital calipers rounding to the nearest 0.1 mm.Data of morphometric characters were presented as percentages or ratios of standard length (SL) and head length (HL), except where otherwise indicated.Terminology of osteology follow Amaoka (1969).The distribution map was generated from Ocean Data View (Schlitzer, 2023).

Methods for taking counts and measurements follow
Specimens are deposited in the Pisces Collection of the National Museum of Marine Biology and Aquarium , Taiwan (NMMB-P).Counts and measurements of 13 specimens of P. taiwanensis, including one dextral and 12 sinistral specimens were taken.The dextral specimen (NMMB-P 38889) and one sinistral specimen (NMMB-P 38874) were dissected to examine the internal organs.Determination of sex follow Amaoka & Ho (2019), with specimens possess rostral spine, mandibular knob, and a rather wide interorbital space identified as male, others as female or juvenile.

Ethical statements
All materials used were either museum collections or collected by us from local fish markets and were dead when obtained.No living animals were used in this study.

Specimen examined
Dextral: NMMB-P 38889 (1 male, 95.2 mm SL), off Ke-Tzu-Liao fishing port (ca.22 • 42 53 N, 120  and 3. Dorsal-fin rays 103.Ocular-side pectoral-fin rays 13; blind-side pectoral-fin rays 10.Ocular-side pelvic-fin rays 6; blind-side pelvic-fin rays 6. Anal-fin rays 81.Caudal-fin rays   Body elliptical, greatest depth 2.3 in SL; both dorsal and ventral profiles of body convex and nearly asymmetric.Head moderate, length 4.1 in SL; anterior profile of head steep, and with deep concavity in front of interorbital space.Snout short, length 5.4 in HL, distinctly shorter than eye diameter.Rostral spine present but blunt.Eyes small, upper-eye diameter 3.0 in HL, and lower-eye diameter 3.2 in SL; lower eye slightly in advance of upper eye.Orbital spines absent.Interorbital space concave, its width 8.1 in HL.Two nostrils present on ocular side and situated anterior to lower eye; anterior nostril forming tube and with small flap; posterior nostril without flap, situated immediately in front of lower eye; nostrils on blind side present, situated below dorsal-fin origin.
Mouth large, oblique; upper-jaw length on ocular side 2.9 in HL; its anterior tip at same vertical through tip of lower jaw; its posterior tip at same vertical through anterior margin of upper eye.Upper-jaw teeth on both ocular and blind sides biserial at anterior half, those on outer row rather stouter; lower-jaw teeth on both ocular and blind sides uniserial, slightly larger and more-spaced than those on upper jaw.
Gill rakers present on outer-four arches; those on first arch short, laterally compressed; first raker on lower arch with several spinules on inner face, while others sooth, not serrated; those on second and third arches about same size as those on first arch; rakers on fourth arch distinctly shorter; no rakers on upper limb of all four Scales on ocular side present and deciduous; scales absent on snout and both ocular-side jaws; scales on blind side deciduous.
Dorsal-fin origin before midline of interorbital space; anterior most rays not elongated; longest ray at slightly posterior to middle of body.Anal-fin origin at same vertical through base of ocular-side pectoral fin; its outer margin subsymmetrical to dorsal-fin margin.Membranes of dorsal and anal fins perforated at base.Pectoral fins on both ocular and blind sides short, length 1.5 and 2.6 in HL, respectively.Origin of ocular-side pelvic fin at tip of isthmus; origin of blind-side pelvic fin opposite to fourth ray of ocular side.Tip of isthmus at same vertical through posterior margin of lower eye.Caudal fin slightly pointed.All rays simple except for middle 13 caudal-fin rays.

Coloration
When fresh (Fig. 1A), body light brown, with abdominal region dusky.All fin rays slightly darker than body color.When preserved (Fig. 1B), body coloration similar to fresh but slightly yellowish; all fin rays dusky; peritoneum scattered with dense black pigments.

Osteology
Neural spines absent on first vertebra; parapophysis well-developed on fifth to tenth vertebra; pleural ribs and epipleurals absent (Fig. 1C).Four plates on caudal skeleton, including three hypurals and parhypural; epural fused with second uroneural and sixth hypural; urostyle fused with fourth to fifth hypurals; second and third hypurals fused.Distal margin of all hypural plates unbranched and no distinct clefts or grooves.

Visceral organs
Anus situated on blind side and immediately anterior to first anal-fin ray; genital papilla situated on ocular side, slightly anterior to anus (Fig. 2).Liver situated on ocular side of abdominal cavity; stomach and intestine coils on blind side of abdominal cavity.

Remarks
The present specimen is identified as Parabothus taiwanensis (Amaoka & Shen, 1993) in having 103 dorsal-fin rays; 81 anal-fin rays; 60 lateral-line scales; a short and blunt rostral spine present in male; biserial upper-jaw teeth; and perforations present on dorsal and anal fins (Voronina & Causse, 2014;Amaoka & Ho, 2019).Moreover, the meristic and morphometric characters of the dextral specimen fall in the range of other sinistral specimens (Tables 2 and 3).
Despite the position of the eyes, our dextral specimen possesses several characteristics that are also reversed compared to normal specimens (Figs.

DISCUSSION
Several cases (mainly Paralichthys spp.) showed that there are higher incidences of reversal in cultured flounders than in the wild populations, and the reasons for this phenomenon still remain unknown (Schreiber, 2013).However, it was suggested several environmental parameters may affect the selection and frequency of sinistral and dextral forms (Schreiber, 2013).With the extremely rare cases (only 2 specimens) in bothid species, it is difficult to explain whether the reversed forms are naturally rare or have been selected in the natural environment.

The first case of situs inversus viscerum in Bothidae
Situs inversus viscerum was only found in a few flatfish species and this was not found in the first reversed bothid, Chascanopsetta lugubris, i.e., internal organs not reversed, according to Kuroshima, Obata & Kawai (2022).In contrast, situs inversus viscerum is present in our specimen, which is the first case in the family Bothidae.In normal individuals, the liver is on the left side of the abdominal cavity, whereas the stomach and intestine coils are on the right side (Amaoka, 1964), however, the reversed specimen has its liver, stomach, and intestine coils on the opposite side of sinistral specimens.Hashimoto et al. (2002) isolated a reversed clonal line of homozygous Japanese flounder (Paralichthys olivaceus) through gynogenesis, and found that the reversed individuals have higher frequency of the reversal of visceral organs than normal individuals.Moreover, they found that the reversal of the visceral organs was not always accompanied by the reversal Based on the aforementioned results, we may conclude that although the situs inversus viscerum may have a higher chance in reversed flatfishes.However, the mechanism still remains unknown, and it appears to be a somewhat random condition among flatfishes.